Lactobacillus rhamnosus JY02 Ameliorates Sarcopenia by Anti-Atrophic Effects in a Dexamethasone-Induced Cellular and Murine Model

Sarcopenia is defined as loss of muscle mass and strength due to aging. Recent studies show that sarcopenia may improve via the gut–muscle axis, suggesting that gut health may affect muscle phenotypes. In this study, we aimed to investigate the ability of Lactobacillus rhamnosus JY02 as a probiotic strain isolated from kimchi to alleviate sarcopenia. L. rhamnosus JY02-conditioned medium (CM) reduced dexamethasone (DEX)-induced myotube diameter atrophy and expression of muscle degradation markers (MuRF1 and atrogin-1) in C2C12 cells. The amelioration of sarcopenia was investigated by measuring body composition (lean mass), hand grip strength, myofibril size (using histological analysis), and mRNA and protein expression of muscle-related factors in a DEX-induced mouse model. The results of these analyses showed that L. rhamnosus JY02 supplementation promoted the production of muscle-enhancement markers (MHC Iβ, MHC IIα, and Myo-D) and reduced both the production of muscle degradation markers and the symptoms of muscle atrophy (loss of lean mass and muscle strength). We also found decreased levels of pro-inflammatory cytokines (IL-6, IFN-γ) and increased levels of anti-inflammatory cytokines (IL-10) in the serum of DEX+JY02-administered mice compared to those in DEX-treated mice. Overall, these results suggest that L. rhamnosus JY02 is a potent probiotic supplement that prevents sarcopenia by suppressing muscle atrophy.


Acid tolerance, Bile acid Tolerance, and Intestinal Adhesion Capacity
To reflect the acidic conditions of the stomach, MRS broth was adjusted to pH 2.5 using 6N HCl. After autoclaving, porcine gastric mucosa-derived pepsin (Sigma-Aldrich, USA) was added to a final concentration of 1,000 units/mL and sterilized using a membrane syringe filter having a pore size of 0.45 μm. To evaluate the acid resistance of the strain, 100 μL (1%) of the strain culture was inoculated into 10 mL of an acidic solution and incubated at 37°C for 2h. The culture media was spread on an MRS agar plate to measure the number of viable cells, and the number of viable cells incubated for 0 h was compared with the 2 hours incubated viable cells as a control.
To form an artificial bile acid conditioned medium, 0.5% oxgall was added to MRS Broth and used after autoclaving. 100 μL (1%) of lactic acid bacteria inoculated into 10ml of MRS broth added 0.5% oxgall and incubated for 24 h. After transferring the culture medium to a 96-well plate, absorbance was measured at the wavelength of 600 nm. Bile acid resistance was expressed as a growth percentage by comparing the absorbance at 0 h and at 24 h.
HT-29 cells were cultured in RPMI (Hyclone) containing 10% fetal bovine serum (FBS-Heat-Inactivated; #S 101-07, welgene), 1% Non-Essential amino acid (MEM NEAA; Gibco) and 1% Anti-anti (Antibiotic; Gibco) in a 37°C with 5% CO₂ incubator. to confirm the ability to adhere to HT-29 cells, HT-29 cells were seeded into a 24-well plate at a concentration of 4×10 5 /cm 2 and used when confluency reached 90%. 500 μL of 10X concentrated lactic acid bacteria were resuspended into 500 μL of pure RPMI medium (without 1% antibiotic) and added in each well and plates were incubated for 2 h at 37°C, 5% CO 2 conditions. After incubation, plates were washed three times with PBS while stirring at a speed of 200 rpm for 3 minutes each to remove non-adhered lactic acid bacteria. Cells isolated by trypsinization were diluted with 0.1% peptone water, spread on MRS Agar plates, incubated at 37°C for 48 hours, and viable cells were counted.

Measurement of GFP, Life Span Assay, and Movement Assay Using C. elegans
C. elegans were synchronized and placed onto Nematode Growth Medium (NGM) Agar plates seeded with concentrated Escherichia coli OP50 and incubated to the L4 stage at 25°C.
For life span assay, CF512 [fer-15(b26);fem-1(hc17)] mutants and PD4251 (ccIs4251 and myo-3::GFP) mutants were used each experiments. The L4 state of C. elegans were transferred one by one using a platinum wire to the NGM plates about 30 animals. The viability was determined by transferring the worms one by one to a new plate until all worms were dead and counting under a microscope.
For movement assay, PD4251 in the L4 state was exposed to the experimental strain and then transferred to a fresh plate. On the 5th and 8th days after exposure, the movements of 3 randomly selected worms were recorded for 20 seconds. A score of 3 points was given for S-shaped movements in which both the pharynx and tail moved, 2 points for motility in which only the front part of the body moved, and 1 point for motility in which only the pharynx moved without movement.

Evaluation of Potential Probiotics Properties of Kimchi-Derived LAB
According to morphological and biochemical characterization, 246 strains showing Gram-positive, catalase, and KOH-negative were finally isolated (Table S1). Also, JY02 was included in lactic acid bacteria isolated from Mustard leaf kimchi.
To investigate the probiotics characteristics of the isolated lactic acid bacteria, acid tolerance, bile acid tolerance, and intestinal adhesion experiments were conducted. Also, Lactobacillus rhamnosus GG (LGG), well known as a probiotic strain, was used as a positive control. As a result of measuring the acid tolerance and bile acid tolerance of lactic acid bacteria under acidic (pH 2.5) and bile acid (0.5% oxgall) conditions, JY02 (Named as LFR20-030 in the graph) showed an excellent survival rate of more than 80% against acid resistance (Fig. S1A) and showed higher bile resistance than positive control LGG (Fig. S1B).
As a result of evaluating the adhesion ability of the strain, JY02 showed 100% adhesion rate similar to that of LGG (Fig. S1C). It was confirmed that JY02 has a probiotic effect through the evaluation of acid tolerance, bile acid tolerance, and adhesion ability to the intestine.

Functional Screening Using the C. elegans Model.
For functional screening, an innate immunity experiment was conducted using the AY102 mutant, in which the intensity of GFP combined with the pmk-1 gene increases when immunity increases, and a life span assay was conducted to confirm whether there is an effect of extending lifespan through anti-aging effect. JY02 showed a high expression of pmk-1 (Fig.   S2A) and showed a significant life span extension effect (Fig. S2B). Fluorescence expression of the myo-3 gene was evaluated on the 7th day after exposure to the experimental bacteria using PD4251 mutant, in which GFP is conjugated to the myosin heavy chain gene that activates cytoskeletal motor activity in the body wall muscles and vulval muscles.
As a result of quantifying the expression of the myo-3 promoter in body wall muscle as the area of muscle nucleus or the amount of GFP expression per area, JY02 showed significantly wider and higher myo-3::GFP expression compared to OP50 control (Fig. S3A).
JY02 also significantly extended the life span of PD4251 (Fig. S3B). In a movement experiment to evaluate whether it is effective in improving the movement that decreases over time, it was confirmed that the movement of JY02 remained high on the 8th day (Fig. S3C).
These results suggest the potential of JY02 to alleviate or delay age-related muscle decline.
JY02, which improved age-related movement, was finally selected and confirmed to belong to the genus L. rhamnosus through 99% (1494/1509) identical to the 16s rRNA gene of L. rhamnosus strain JCM1136 (accession No: NR_043408). 16s rRNA sequence from isolated JY02 was shown in Table S2 and the phylogenetic tree shown in Fig. S4.

Mice.
We assessed the expression levels of five cytokines (IL-6, IFN-γ, IL-10, TNF-α, and IL-12p70) and chemokines of MCP-1 in mouse serum to determine the anti-inflammatory effects of JY02 on DEX-induced muscle atrophy. As shown in Fig S5, JY02 pretreatment decreased pro-inflammatory factors levels (IL-6, IFN-γ, and MCP-1) and enhanced levels of IL-10 compared with the DEX-treated group. However, there was no significant difference in serum levels of two cytokines (TNF-α, and IL-12p70) between the normal and DEX-treated groups ( Fig. S5E and F).

Supplementary Figure 1. Evaluation of Acid Resistance, Bile Acid Resistance, and
Intestinal Adhesion of LAB (A) Survival rate of strains exposed for 2 hours to gastric juices containing pepsin at pH 2.5 compared to positive control LGG (100%). Survival (%) = 2 h viable cell count / 0 h viable cell count × 100%. (B) Survival rate of strains exposed for 24 h to bile acid condition (A) Analysis of pmk-1::GFP fluorescence intensity in AY102 [vha-6p::pmk-1::GFP + rol-6(su1006)] using the fluorescence microscopy (IX53, Olympus, Japan). The intensity was measured using ImageJ and divided by the number of worms. E. coli OP50 was used as a negative control and LGG as a positive control. (B) L4 state of CF512 [fer-15(b26);fem-1(hc17)] were exposed to lactic acid bacteria until death, the number of deaths by day is counted. Statistics were calculated relative to worms exposed to E. coli OP50. Data were performed in 3 repetitions (n = 30 per plate). * p < 0.01 vs. the control group (OP50).
(A) Analysis of myo-3::GFP fluorescence intensity in PD4251. Fluorescence expression was measured on the 7th day after exposing worms in the L4 state to lactic acid bacteria.
Photographs observed by fluorescence microscopy (IX53, Olympus, Japan). The intensity was measured using the ImageJ program. (B) L4 state of PD4251 was exposed to lactic acid bacteria until death, the number of deaths by day is counted. Statistics were calculated relative to worms exposed to E. coli OP50. Data were performed in 3 repetitions (n = 30 per plate). (C) Motion score of C. elegans on day 5 and day 8 of exposure to probiotics. * p < 0.01 vs. the control group (OP50).

Supplementary Figure 4. The phylogenetic position of JY02.
Phylogenic neighbor-joining JY02 by 16S rRNA sequence. JY02 is named as LFR20-030 in the graph.